Cumulus Cells Significantly Reduce Hydrogen Peroxide Induced Damage to Metaphase-II Mouse Oocyte Spindle and Chromosomal Alignment Faten Shaeib1, Jashoman Banerjee1, Mili Thakur1, Mohammed Saed1, Awoniyi Awonuga1, Michael Diamond1, Ghassan Saed1, and Husam Abu-Soud1 1Wayne State University, United States Oxidative stress is known to deteriorate oocyte quality by inducing structural damage to the meiotic spindle (MT) and chromosomal alignment (CH). the presence of surrounding intact cumulus cells, the distance of cumulus to oocyte, active gap junction proteins connexin 43, and sufficient quantities of antioxidant are vital in preventing oxidative damage to the oocyte. Here, we investigate the protective capacity of the cumulus cells against H2O2 damage. Metaphase-II oocytes with and without cumulus cells were exposed to various H2O2 concentrations for 30 min, at 37°C, fixed, stained, imaged by confocal microscope, and scored for MT and CH changes as well as evaluated for connexin 43. Good spindle configuration (score 1, 2), was for organized microtubules in a barrel-shaped, whereas, abnormal or poor (score 3, 4) was for spindle length reduction, disorganization and complete absence spindle. Chromosomal configuration was considered as good (score 1, 2) when chromosomes were normally arranged at the equator of the spindle, while poor (score 3, 4) when the chromosomes were dispersed or showed aberrant or less condensed appearance. At low levels of H2O2 and CH scores for oocytes without cumulus cells were significantly higher than with cumulus cells (p<0.001). At high levels of H2O2 (>25 μM) the poor MT and CH scores were not different between groups. Additionally, catalase (H2O2 consumer) mRNA levels were significantly higher in oocytes with cumulus cells compared to those without (p<0.001). the density and the volume of connexin 43 were significantly reduced in H2O2 (100 μM) treated oocytes with cumulus cells as compared to untreated (p<0.001). Thus at low levels of H2O2, cumulus cells significantly damage to metaphase II oocyte spindle and chromosomal alignment, whereas at high levels it fails to do so due to the detachment of cumulus cells.

Oxidative Stress Induces Metaphase II Mouse Oocyte Spindle Damage Faten Shaeib1 and Husam Abu-Soud1 1Wayne State University, United States Reactive oxygen species such as peroxynitrite (ONOO ), hypochlorous acid (HOCl), hydroxyl radical (HO ), or hydrogen peroxide (H2O2) has been shown to mediate damage of metaphase II mouse oocyte microtubule (MT) and chromosomal (CH) structure, markers of oocyte quality. the objective of this study was to compare the damaging effect of these ROS on oocyte quality in the presence and absence of surrounding cumulus cells, utilizing 1-4 scoring system to evaluate the changes in MT and CH. Good spindle configuration (score 1, 2), was for organized microtubules in a barrel-shaped, whereas, abnormal or poor (score 3, 4) was for spindle length reduction, disorganization and complete absence spindle. Chromosomal configuration was considered as good (score 1, 2) when chromosomes were normally arranged at the equator of the

spindle, while poor (score 3, 4) when the chromosomes were dispersed or showed aberrant or less condensed appearance. Longest axis; width; volume of both MT and CH also have been evaluated utilizing confocal 3-D microscopy. in all ROS, the plot of poor scoring versus ROS concentrations showed a dose-dependent effect on both spindle structure and CH alignment. in non-cumulus oocyte treatments, the 100% of poor scoring was reached in the following order HO , ONOO , H2O2 and HOCl. in HO and H2O2 treatments, cumulus presence showed gained of protection of oocyte quality, whereas in HOCl and ONOO treatments, no protection was provide by cumulus cells. Treatment with ONOO induced MT and CH length and width shortening, as well as, decrease in MT and CH volumes in a dose dependent manner. on linear regression, with single unit increase in ONOO concentration, there was 0.6 % decrease in Length (p<0.013) and 22.6% decrease in volume (p<0.007). the nature of the environments that generated ROS has a different impact on the oocyte quality.

Melatonin Prevents Myeloperoxidase Heme Destruction and the Generation of Free Iron Mediated by Self-Generated Hypochlorous Acid Faten Shaeib1, Dhiman Maitra1, Jashoman Banerjee1, Ibrahim Abdulhamid1, Michael Diamond1, Ghassan Saed1, Subramaniam Pennathur2, and Husam Abu-Soud1 1Wayne State University, United States, 2University of Michigan, United States Recently, we have shown that myeloperoxidase self-generated hypochlorous acid (HOCl) formed during catalysis is able to destroy the enzyme heme moiety, and serve as a major source of free iron. the toxicity of free iron is exerted by generation of free radicals through its participation in the Fenton reaction. Utilizing UV-visible spectroscopy, hydrogen peroxide (H2O2)-specific electrode measurements, and ferrozine assay, here, we show that melatonin prevents MPO heme destruction and subsequent iron release mediated by self-generated HOCl. Melatonin inhibits MPO activity by: 1) serving as a one electron substrate of the higher MPO oxidative states intermediates, Compounds I and II, that are formed through the reaction of H2O2/HOCl with MPO, allowing the enzyme to oxidize melatonin as a one-electron transfer instead of generating HOCl; 2) binding to MPO above the heme iron or in the entrance of the MPO heme pocket thereby preventing the access of H2O2 to the catalytic site of the enzyme; and/or 3) direct scavenging of HOCl. Collectively, in addition to acting as an antioxidant and MPO inhibitor, melatonin can exert its protective effect by preventing the release of free iron mediated by self-generated HOCl. Our work may establish a direct mechanistic link by which between melatonin exerts its protective effect in chronic inflammatory diseases with MPO elevation.

doi: 10.1016/j.freeradbiomed.2013.10.724

doi: 10.1016/j.freeradbiomed.2013.10.725

doi: 10.1016/j.freeradbiomed.2013.10.726